microrna-140 plays dual roles in both cartilage development and...

MicroRNA-140 plays dual roles in bothcartilage development and homeostasis

Shigeru Miyaki,1,4 Tempei Sato,2,4 Atsushi Inoue,2 Shuhei Otsuki,1 Yoshiaki Ito,2

Shigetoshi Yokoyama,2 Yoshio Kato,3 Fuko Takemoto,2 Tomoyuki Nakasa,2 Satoshi Yamashita,2

Shuji Takada,2 Martin K. Lotz,1 Hiroe Ueno-Kudo,2 and Hiroshi Asahara1,2,5

1Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA;2Department of Systems Biomedicine, National Research Institute for Child Health and Development, Tokyo 157-8535, Japan;3Biomedical Research Institute for Cell Engineering (RICE), National Institute of Advanced Industrial Science and Technology(AIST), Tsukuba 305-8562, Japan

Osteoarthritis (OA), the most prevalent aging-related joint disease, is characterized by insufficient extracellularmatrix synthesis and articular cartilage degradation, mediated by several proteinases, including Adamts-5. miR-140is one of a very limited number of noncoding microRNAs (miRNAs) specifically expressed in cartilage; however,its role in development and/or tissue maintenance is largely uncharacterized. To examine miR-140 function intissue development and homeostasis, we generated a mouse line through a targeted deletion of miR-140. miR-140�/�

mice manifested a mild skeletal phenotype with a short stature, although the structure of the articular joint cartilageappeared grossly normal in 1-mo-old miR-140�/� mice. Interestingly, miR-140�/� mice showed age-related OA-likechanges characterized by proteoglycan loss and fibrillation of articular cartilage. Conversely, transgenic (TG) miceoverexpressing miR-140 in cartilage were resistant to antigen-induced arthritis. OA-like changes in miR-140-deficient mice can be attributed, in part, to elevated Adamts-5 expression, regulated directly by miR-140. We showthat miR-140 regulates cartilage development and homeostasis, and its loss contributes to the development ofage-related OA-like changes.

[Keywords: MicroRNA; miR-140; osteoarthritis; cartilage; Adamts-5]

Supplemental material is available at http://www.genesdev.org.

Received February 10, 2010; revised version accepted April 13, 2010.

MicroRNAs (miRNAs) are a class of noncoding RNAsthat negatively regulate genes involved in numerous bio-logical processes. Hundreds of miRNAs have been iden-tified in various organisms, and many are evolutionarilyconserved. Moreover, an estimated one-third of all mam-malian mRNAs are regulated by miRNAs, demonstratingthe essential role of miRNAs in controlling gene expres-sion (Lewis et al. 2005). miRNAs are generated as longprimary transcripts (pri-miRNAs) that are processed bythe enzymes Drosha and Dicer into short ribonucleotides(;22 nucleotides long). The mature miRNAs are thenincorporated into the RNA-induced silencing complex(RISC). The miRNA–RISC complex mediates the degra-dation of specific mRNA targets and/or the repression ofmRNA translation via interactions with the 39 untrans-lated regions (UTRs) that are partially sequence-specific(Bartel 2004). Several miRNAs exhibit a tissue-specific ordevelopmental stage-specific expression pattern and have

been associated with human diseases such as heart disease(van Rooij et al. 2006) and arthritis (Iliopoulos et al. 2008;Nakasa et al. 2008; Stanczyk et al. 2008; Yamasaki et al.2009). In addition, mice with limb- or cartilage-specificdeletion of the miRNA-processing enzyme Dicer exhibiteda severe phenotype with reduced limb size but normalpatterning (Harfe et al. 2005; Kobayashi et al. 2008). Dicer isindispensable for mature, functional miRNAs; therefore,this finding suggests that miRNAs play a critical role inskeletal development.

Recent studies have revealed the cartilage-specific ex-pression of miR-140 in mouse embryos and zebrafish(Wienholds et al. 2005; Tuddenham et al. 2006). Using anmiRNA microarray, we demonstrated considerable miRNAexpression differences between human articular chondro-cytes and mesenchymal stem cells. Furthermore, miR-140exhibited the largest expression difference between the twocell types (Miyaki et al. 2009). The study of miR-140 maythus be the key miRNA to open a new insight of cartilagebiology. However, the function of miRNAs in articularcartilage has not been analyzed by targeted deletion.

Previous studies also found reduced miR-140 expres-sion in human osteoarthritis (OA) cartilage (Iliopoulos

4These authors contributed equally to this work.5Corresponding author.E-MAIL [email protected]; FAX (858) 784-2695.Article published online ahead of print. Article and publication date areonline at http://www.genesdev.org/cgi/doi/10.1101/gad.1915510.

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et al. 2008; Miyaki et al. 2009), which may contribute tothe abnormal gene expression pattern characteristic ofOA. These findings prompted us to examine the role ofmiR-140 in cartilage development and homeostasis bygenerating miR-140-deficient mice and cartilage-specificmiR-140 transgenic (TG) mice. The results indicate thatmiR-140 plays dual roles in both cartilage developmentand homeostasis, in part via regulating Adamts-5, a majorcartilage matrix-degrading protease in OA (Glasson et al.2005; Stanton et al. 2005).

Results

Targeted deletion of cartilage-specific miR-140

To define the in vivo function of miR-140, we targeted themouse miR-140 sequence for deletion. We deleted theregion containing miR-140 in the intron between exons16 and 17 of the WW domain-containing E3 ubiquitinprotein ligase 2 (Wwp2), and inserted a neomycin (neo)resistance cassette flanked by loxP sites (Fig. 1A,B). Thefloxed phosphoglycerine kinase (PGK)-neo cassette wasremoved by crossing with Meox-Cre TG mice, and Cre-mediated neo excision was confirmed by genomic PCR(Fig. 1C). Quantitative real-time PCR (qPCR) analysis ofchondrocyte RNA showed a complete absence of miR-140 in miR-140�/� mice (Fig. 1D). We confirmed that theWwp2 protein levels were unchanged in miR-140�/�

mice (Fig. 1E).

Skeletal growth in miR-140�/� mice

miR-140�/� mice were born in normal Mendelian ratiosand were fertile. Skeletal development during embryo-genesis in miR-140�/�mice appeared grossly normal (Fig.2A). Postnatally, miR-140�/� mice manifested a mildskeletal phenotype, with short stature and low bodyweight (Fig. 2B–D), as well as craniofacial deformitiescharacterized by a short snout and domed skull (Fig. 2E).This craniofacial phenotype in miR-140�/� mice showspartial similarity to that of cartilage-specific Dicer-deficientmice (Kobayashi et al. 2008), supporting the notion thatmiR-140 is a tissue-specific miRNA important in cartilagedevelopment. As miR-140 is expressed in cartilage and notin other tissues by whole-mount in situ hybridization(Wienholds et al. 2005; Tuddenham et al. 2006), reducedskeletal tissues of miR-140�/� mice may account for thisweight loss phenotype.

The hind-limb bones were shorter in miR-140�/� micecompared with wild-type mice (Fig. 3A). In the growthplates of wild-type mice, miR-140 was expressed in pro-liferating chondrocytes but not in hypertrophic chondro-cytes that expressed Col10a1 (Fig. 3B). The width of thetibial growth plates in 1-mo-old mice was reduced in miR-140�/� mice compared with wild-type mice (Fig. 3C). Thenumber of proliferating chondrocytes was significantlydecreased at postnatal day 10 (P10) in miR-140�/� mousegrowth plates (Fig. 3D,E), whereas no significant changeswere observed in hypertorophyic zones (Fig. 3C). Thesefindings indicate that the mild skeletal phenotype in miR-

140�/�mice with short stature results from a reduction inproliferating chondrocytes.

OA-like pathology in miR-140�/� mouse knee joints

Chondrocytes play a critical role not only in skeletaldevelopment, but also in articular cartilage formationand maintenance. Analysis of pri-miR-140 expression in2-mo-old wild-type mice by in situ hybridization showedmiR-140 expression in chondrocytes from the surface tomiddle zones of articular cartilage and in the menisci,overlapping with expression of the chondrocyte markerCol2a1 (Fig. 4A). Furthermore, the structure and shapeof knee joints—including articular cartilage, menisci,and ligaments—were analyzed by Safranin O staining andthree-dimensional (3D) computed tomography (CT). Theyall appeared to be normal in miR-140�/� mice at birth and1 mo of age (Supplemental Fig. S1).

Because miR-140 expression was shown to be reducedin human OA cartilage (Iliopoulos et al. 2008; Miyakiet al. 2009), we examined the potential role of miR-140 incartilage homeostasis. OA initiation and progression ismediated by various stimuli and circumstances, includingthe following three main factors: age-related changes inhomeostatic balance, excessive mechanical stress some-times triggered by joint injury, and transient inflammationin the articular joint damaging the cartilage matrix. There-fore, to determine the potential role of miR-140 in cartilagehomeostasis, we used three different animal models of OA:an aging model, a surgical model, and an antigen-inducedarthritis (AIA) model.

First, we tested whether loss of miR-140 affected age-related onset of OA changes, and observed that miR-140�/� mice developed an age-related OA-like pathology.Knee joints from 3-mo-old mice showed reduced SafraninO staining in femoral condyles and tibial plateaus, in-dicative of proteoglycan loss (Fig. 4B). By 8 mo, overt car-tilage degradation was apparent as more severe proteo-glycan loss, a roughened articular surface, and fibrillation;these changes were not observed in age-matched wild-type mice (Fig. 4B). By 12 mo, miR-140�/� mice showedsevere structural cartilage defects (Fig. 4B) that were notassociated with synovial hyperplasia. We also observedOA-like changes in elbow and ankle joint articular car-tilage of miR-140�/� mice at 12 mo old, compared witharticular cartilage from wild-type mice (SupplementalFig. S2). To quantify OA-like pathological changes inthe articular cartilage, an OA scoring system was usedto validate several aspects of the histological changes(Chambers et al. 2001; Glasson et al. 2004). OA scoreswere significantly higher in miR-140�/� mice comparedwith wild-type mice (Fig. 4C). These results support thehypothesis that miR-140 is a critical regulator of cartilagehomeostasis, and its loss contributes to cartilage degra-dation characteristic of OA.

Next, we used the surgical arthritis model, in which thearticular cartilage was exposed to an excessive mechanicalload, due to joint instability caused by surgical resectionof the medial meniscotibial ligament (MMTL) (Glassonet al. 2007). Consistent with observations in the aging OAmodel, the surgical arthritis model also demonstrated

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that miR-140�/� mice exhibit accelerated proteoglycanloss and fibrillation of articular cartilage in knee jointscompared with the wild-type mice at 8 wk after surgery,which is reflected in higher OA scores (Fig. 4D,E).

Cartilage-specific miR-140 TG mice exhibitedresistance to AIA

We further examined the role of miR-140 in articularcartilage by using the AIA model, in which transient

inflammation was induced by antigen injection. Inflam-matory signals such as interleukin-1b (IL-1b) from thechondrocytes and synovial membrane of articular jointspaces mediate OA progression, in part by up-regulatingthe expression of cartilage matrix degradation enzymesin chondrocytes (Goldring and Goldring 2007). In thismodel, as well as in human OA pathogenesis, transientjoint inflammation causes cartilage damage by the in-duction of cartilage-degrading enzymes. The AIA modelis advantageous in that it leads to cartilage damage in a

Figure 1. Generation of miR-140-null mice. (A) Targeting of miR-140. The targeting vector was constructed to replace the endogenouspri-miR-140 locus with a PGK-neo cassette by homologous recombination. The 59 probe and 39 probe used for Southern blot and PCRprimers used for genotyping are indicated. (E) EcoRV, (S) SphI. (B) Southern blot analysis of wild-type (Wild) and miR-140 mutant mice.Genomic DNA was digested with EcoRV and probed with the indicated 39 probe. (C) Confirmation of Cre-mediated neo excision bygenomic PCR. The floxed PGK-neo cassette was removed by crossing with Meox-Cre TG mice. (D) Expression of mature miR-140 inwild-type (Wild) and miR-140�/� mouse embryos at E11.5 as assessed by qPCR. Mature miR-140 was not detected in miR-140�/�mice.Data are expressed as mean 6 SEM (n = 3). (E) Western blot analysis of Wwp2. Wwp2 expression was not detectably different in miR-140�/� compared with wild-type mice.

The roles of miR-140 in cartilage homeostasis

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short time in wild-type mice, which enables us to moni-tor the beneficial effects of miR-140 against cartilage de-gradation by a gain-of-function approach.

Accordingly, we generated cartilage-specific TG mice inwhichmiR-140expressionwasdrivenbyawell-characterized

Col2a1 enhancer sequence (Fig. 5A; Zhou et al. 1995;Krebsbach et al. 1996). We obtained three lines of TGmice and observed up-regulation of miR-140 in cartilageof all three lines (Fig. 5B). These TG mice did not showany apparent abnormalities in skeletal development (data

Figure 2. Growth retardation in miR-140�/�mice. (A) Skeletal preparation with Alcian blue/Alizarin red staining of wild-type (Wild) andmiR-140�/� littermates at E17.5. miR-140�/� embryos exhibited no overt abnormalities in skeletal development. (Left) Whole-mount.(Middle) Dorsal view. (Right) Forelimb. (B) Appearance of wild-type and miR-140�/� mice at 4 wk. Growth retardation wasobserved in miR-140�/�mice. (C) Wild-type and miR-140�/�mice were scored for tail length and body weight at various postnatal stages.miR-140�/� mice showed growth retardation 1 wk after birth (tail length, P < 0.01; body weight, P < 0.05). Data are expressed as mean 6

SEM (n = 5–12). (D) No gross skeletal changes were detectable by X-ray in 3-mo-old mice. (E) Skulls of wild-type and miR-140�/� micestained with Alcian blue/Alizarin red. Dorsal views are shown. miR-140�/� mice exhibited craniofacial bone defects characterized byshort nasal bone, short maxilla, and domed skull. (NC) Nasal capsule, (N) nasal bone, (F) frontal bone, (P) parietal bone, (IP) interparietalbone, (S) supraoccipital bone.

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not shown). To examine whether the miR-140 level inarticular chondrocytes affects cartilage sensitivity to ex-perimental challenge, we assessed AIA in knee joints ofmiR-140 TG mice, miR-140�/� mice, and wild-type mice.We observed similar levels of synovial hyperplasia amongmiR-140 TG mice, miR-140�/� mice, and wild-type mice;however, miR-140�/� mice showed reduced Safranin Ostaining (Fig. 5C). Importantly, miR-140 TG mice wereresistant to proteoglycan and type II collagen loss com-pared with wild-type mice. To quantify the extent ofmatrix degradation, we used the Mankin scoring system(Mankin 1971; Zemmyo et al. 2003) for proteoglycan loss.Mankin scores were significantly lower in miR-140 TG

mice and significantly higher in miR-140�/� mice com-pared with wild-type mice (Fig. 5D). These findings areconsistent with the idea that miR-140 protects against OAprogression.

ADAMTS-5 is a direct target of miR-140 and mediatesOA pathogenesis

Identification of miR-140 target genes can provide newinsights into miR-140 function and OA pathogenesis. Toachieve this goal, we applied DNA array analysis toidentify miRNA targets (Lim et al. 2005; Valencia-Sanchezet al. 2006). Based on computationally predicted target

Figure 3. Skeletal growth in miR-140�/� mice. (A, left) Length of femur. (Right) Long bones were significantly shorter in the miR-140�/� mice. Data are expressed as mean 6 SEM (n = 3). (**) P < 0.01. (B) Expression of pri-miR-140, Col2a1, and Col10a1 in the tibialgrowth plates of P10 mice. miR-140 was detected in proliferating chondrocytes. (Blue bar) Proliferative zone, (red bar) prehypertrophiczone, (green bar) hypertrophic zone. (C) Safranin O staining of the tibial growth plates in 1-mo-old mice. The proliferating zone wasreduced in miR-140�/� mice compared with wild-type mice (Wild). (D,E) BrdU-positive cells were significantly reduced in miR-140�/�

mice at P10. BrdU (green) and DAPI (blue) staining indicate nuclei. Data are expressed as mean 6 SEM (n = 3). (**) P < 0.01.






genes found in the online database TargetScan (http://www.targetscan.org), 17 of the mRNAs in the array thatwere increased in miR-140�/� chondrocytes exhibited con-served miR-140-binding sites in their 39 UTRs with 7-meror 8-mer seeds (Supplemental Table S1). Among the mostoverexpressed was the gene for ADAMTS-5, which de-grades aggrecan and is a critical enzyme for OA pathogen-esis (Glasson et al. 2005; Stanton et al. 2005). We analyzedAdamts-5 sequences in humans, mice, rats, and dogs andobserved highly conserved miR-140-binding sites with8-mer seeds in the Adamts-5 39 UTR (Fig. 6A). Adamts-5expression was significantly increased in chondrocytesfrom miR-140�/�mice and significantly decreased in thosefrom miR-140 TG mice compared with wild-type (Fig. 6B).Similarly, increased ADAMTS-5 protein expression in

articular cartilage in miR-140�/� mice at 1 and 3 mo oldwas visualized by immunohistochemistry (Fig. 6C,D; Sup-plemental Fig. S3). To confirm that miR-140 plays a criti-cal role in aggrecanolysis, we quantified proteoglycan lossfrom cartilage in wild-type mice, miR-140 TG mice, andmiR-140�/� mice. Femoral head cartilage explants werecultured with or without IL-1b, which is a potent cata-bolic stimulus of cartilage matrix degradation. Cartilageexplants from miR-140�/� mice showed significantly in-creased proteoglycan release compared with wild-typecartilage (Fig. 6E). In contrast, IL-1b-induced proteoglycanrelease from the cartilage of miR-140 TG mice was signif-icantly lower compared with wild-type cartilage.

We further tested whether miR-140 regulates Adamts-5mRNA in chondrocytes. Treatment of chondrocytes from

Figure 4. miR-140�/� mice exhibit OA-likepathology. (A) Expression of pri-miR-140 (left)and Col2a1 (right) in articular cartilage of theknee joints in 2-mo-old mice. miR-140 wasdetected in chondrocytes from the surface tomiddle zones of articular cartilage. (B) SafraninO staining of the knee joint in 3-mo-old mice(wild-type, n = 6; miR-140�/�, n = 6), 8-mo-oldmice (wild-type, n = 15; miR-140�/�, n = 13), and12-mo-old mice (wild-type, n = 12; miR-140�/�,n = 11). miR-140 deletion caused an early onsetof OA-like changes. (C) Histopathologic scoresof knee joints. OA scores were significantlyincreased in miR-140�/� mice. Data are ex-pressed as mean 6 SEM. (*) P < 0.05; (**) P <

0.01. (D) Surgical OA was induced in 10-wk-oldwild-type and miR-140�/� mice by resecting theMMTL (wild-type mice, n = 8; miR-140�/�

mice, n = 11). The knee joints were harvested8 wk after surgery and stained with SafraninO for histopathologic analysis. (E) miR-140�/�

mice showed significantly increased OA scorescompared with wild-type mice after surgery.Data are expressed as mean 6 SEM. (*) P < 0.05.

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miR-140�/� mice with ds-miR-140 reduced Adamts-5expression (Fig. 6F), supporting the notion that miR-140negatively regulates Adamts-5 mRNA. To determinewhether Adamts-5 is a direct miR-140 target in intactcells, the Adamts-5 39 UTR, which includes a putativemiR-140-binding site, was cloned downstream from theluciferase gene in an expression vector driven by theSV-40 promoter (Adamts-5 39 UTR). Cotransfection ofHEK293T cells with ds-miR-140 significantly reducedluciferase activity in cells transfected with Adamts-539 UTR (Fig. 6G). No changes in luciferase activity wereobserved in cells transfected with the mutated luciferaseexpression vector (Adamts-5 mut 39 UTR) in response to

ds-miR-140 (Fig. 6G). Taken together, these data indicatethat miR-140 directly regulates Adamts-5 expression andproteoglycan loss in articular cartilage.

Discussion

Recent studies have revealed that miRNAs play criticalroles in various biological events, including development,disease, and immunity (Stefani and Slack 2008; Xiao andRajewsky 2009). However, few molecular networks reg-ulated by miRNAs have been characterized in detail, duein part to difficulty in determining the direct targets ofeach miRNA. Tissue-specific miRNAs targeting mice have

Figure 5. Cartilage-specific miR-140 TG mice are resistant to AIA. (A) miR-140 TG mice were generated with a construct using thecartilage-specific Col2a1 promoter. (B) Significantly increased expression of miR-140 in chondrocytes of miR-140 TG mice comparedwith those of wild-type mice was confirmed by qPCR analysis. These data are results from representatives of a single mouse line (n = 3).Data are expressed as mean 6 SEM. (*) P < 0.05. (C) Seven days after induction of AIA, Safranin O staining and immunohistochemistryfor type II collagen shows higher proteoglycan and collagen levels in miR-140 TG mice and lower levels in miR-140�/� mice comparedwith wild type. (D) Mankin score for proteoglycan loss in wild-type mice (n = 5), miR-140 TG mice (n = 6), and miR-140�/�mice (n = 6) 7d after induction of AIA. Data are expressed as mean 6 SEM. (*) P < 0.05; (**) P < 0.01.






been generated to elucidate the role of miRNAs in tissueand organ development, including heart and muscles(van Rooij et al. 2007; Zhao et al. 2007; Liu et al. 2008);

however, so far there is very limited information on thefunction of miRNAs in cartilage/bone development andrelated diseases.

Figure 6. miR-140 regulates Adamts-5 expression. (A) miR-140 targets the Adamts-5 39 UTR. Sequence alignment of a putative miR-140-binding site in the Adamts-5 39 UTR shows a high level of sequence conservation and complementarity with miR-140. (B)Differential Adamts-5 expression in rib chondrocytes from wild-type mice, miR-140 TG mice, and miR-140�/� mice was evaluated byqPCR. Data are expressed as fold differences compared with wild-type chondrocytes (mean 6 SEM; n = 4–5 per genotype). (*) P < 0.05.(C) Histologic analysis of articular cartilage from 3-mo-old mice. Immunostaining showed an increase in ADAMTS-5-positivechondrocytes in miR-140�/�mice. (D) ADAMTS-5-positive cells were detected in wild-type and miR-140�/� cartilage. Knee joints from3-mo-old wild-type (n = 3) and miR-140�/� (n = 3) mice were examined. Data are expressed as mean 6 SEM. (**) P < 0.01. (E) Femoralhead cartilage explants from miR-140�/� mice cultured in DMEM only (n = 13) or DMEM containing IL-1b (n = 6) showed significantlyincreased proteoglycan release compared with wild-type mice in DMEM only (n = 17) or DMEM containing IL-1b (n = 6). IL-1b-inducedproteoglycan release in miR-140 TG mice in medium (n = 12) was significantly lower compared with that of wild-type mice (n = 10).Data are expressed as fold differences compared with wild-type explants in DMEM only (mean 6 SEM). (*) P < 0.05; (**) P < 0.01. (F)Relative Adamts-5 expression in miR-140�/� chondrocytes was determined by qPCR after transfection with ds-miR-140 (black). Dataare expressed as fold differences relative to negative control cells (gray, mean 6 SEM). (*) P < 0.05. (G) Luciferase activity of pLuc2-miR-140 sensor (miR-140 sensor), pLuc2-Adamts-5 39 UTR (Adamts-5 39 UTR), and pLuc2-Adamts-5 39 UTR with mutated miR-140-binding site (Adamts-5 mut 39 UTR). Luciferase activity was determined in HEK293T cells transfected with miR-140 sensor, Adamts-5

39 UTR, or Adamts-5 mut 39 UTR along with a ds-miR-140 (black) or negative control miRNA (gray) (n = 6). Data are expressed asrelative luciferase activity (mean 6 SEM). (*) P < 0.05; (**) P < 0.01.

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Transcription factor SRY-box-containing gene 9 (Sox9)is a master regulator of chondrocyte differentiation (deCrombrugghe et al. 2000). The expression of miR-140shadows Sox9 expression, and the deletion of Sox9 di-minishes miR-140 expression during embryogenesis(S Miyaki and H Asahara, unpubl.), indicating that miR-140 is under Sox9 regulation in chondrocytes. Althoughthe disruption of Sox9 in mice results in a complete lackof cartilage development, miR-140�/� mice showed onlya mild skeletal phenotype, with short stature and cranio-facial changes. Results of studies targeting mice at spe-cific developmental stages show that Sox9 is not onlyessential for chondrogenesis initiation, but is also impor-tant to produce large numbers of proliferating chondro-cytes (Akiyama 2008). In the present study, the expres-sion pattern of miR-140 was detected in the proliferationzone during endochondral ossification, and fewer pro-liferating chondrocytes were observed in miR-140�/�

mice. Thus, regulation of proliferating chondrocytesmay be one of the Sox9 functions mediated by miR-140.

Mice that are Dicer-deficient in cartilage tissues show aprogressive and profound reduction in proliferating growthplate chondrocytes, which leads to severe skeletal growthdefects and premature death (Kobayashi et al. 2008);however, this severe phenotype may reflect the effect ofreducing all miRNAs, including ubiquitous ones. In thisregard, our study provides the first example showing boneand cartilage phenotypes in mice caused by tissue-specificmiRNA.

The phenotype of miR-140�/� mice on skeletal frame-work is not drastic, and the mice can survive for >1 yr;however, this mild phenotype provides us with an oppor-tunity to monitor another aspect of miR-140 function incartilage. Chondrocytes have two major functions: endo-chondral ossification for proper skeletal development, andarticular cartilage maintenance for joint movement. Al-though chondrocytes in endochondral ossification disap-pear after bone development has been completed, articularcartilage chondrocytes persist, secreting the extracellularmatrix that protects tissue from damage. The role ofmiRNAs in tissue homeostasis has not yet been wellelucidated, other than a few studies such as those report-ing the critical role of miR-143/145 in smooth muscle cellmaintenance (Elia et al. 2009).

Articular cartilage is a good tissue for characterizing themolecular network involved in tissue homeostasis. Thecartilage framework consists of a cartilage-specific extra-cellular matrix, which includes type II collagen and pro-teoglycans, and is maintained by chondrocytes embeddedin the matrix. To maintain the integrity of this cartilageframework, the matrix is continuously undergoing remod-eling via matrix-degrading enzymes; this degradation isbalanced by the secretion of newly synthesized matrixproteins. The balance of these catabolic and anabolic sig-nals in cartilage tissue is critical for cartilage homeostasis;aging, inflammation, or injuries may promote catabolicsignals, including ADAMTS-5 and matrix metallopepti-dase-13 (MMP-13), which can lead to arthritis changes. OAis the result of this age-related loss of the homeostaticbalance between cartilage degradation and repair; it is

aggravated by joint inflammation or excessive mechanicalload (Goldring and Goldring 2007; Hashimoto et al. 2008;Goldring and Marcu 2009). Treatment options are limited,and new therapeutic targets that regulate the cartilagehomeostasis balance should be sought.

To determine the role of miR-140 in articular cartilage,we examined three different arthritis models: age-related,surgical, and inflammatory (AIA). Disruption of miR-140in vivo induced the early onset of spontaneous OA-likechanges in articular cartilage of the age-related model andmore severe OA-like changes in the surgical model. Trans-fection of human chondrocytes with ds-miR-140 down-regulated IL-1b-induced Adamts-5 expression (Miyaki et al.2009). Consistent with our findings that miR-140 exertsa protective effect in cartilage, miR-140 TG mice wereresistant to cartilage matrix degradation in the inflamma-tory model, suggesting that the regulation of miR-140 hasimportant implications for drug design in OA treatment.

To address the mechanism of enhanced cartilage degra-dation in miR-140�/� mice, we showed the up-regulationof Adamts-5, a direct target of miR-140. ADAMTS-5 wasshown to be a critical cartilage-degrading enzyme, becauseanimals deficient in ADAMTS-5 activity are resistantto cartilage degeneration in the surgical OA model andinflammatory arthritis model (Glasson et al. 2005; Stantonet al. 2005). ADAMTS-5 also appears to be the majorenzyme responsible for aggrecan degradation in humanOA on the basis of increased mRNA and protein expres-sion in OA cartilage (Malfait et al. 2002). Nevertheless,regulatory mechanisms of Adamts-5 expression have notbeen clearly elucidated. Two groups reported recently thatRunx2 regulates Adamts-5 expression (Thirunavukkarasuet al. 2007), and hedgehog signaling regulates the expres-sion of Adamts-5 via Runx2 (Lin et al. 2009). In addition tothese transcriptional regulations, in the present study wedemonstrated that Adamts-5 expression was tightly regu-lated by miR-140 at the post-transcriptional level.

Although the critical role of miR-140 in cartilage mainte-nance may be explained largely by identifying Adamts-5as its direct target, miRNAs are believed to regulate mul-tiple target mRNAs; therefore, the role of miR-140 incartilage homeostasis may involve the regulation of ad-ditional genes, as listed in Supplemental Table S1. Accord-ingly, gene expression analysis by microarray and qPCRrevealed an up-regulation of other cartilage-related cata-bolic factors, such as matrix degradation enzymes, and adown-regulation of cartilage matrix genes in chondrocytesof miR-140�/� mice as early as P3 (Supplemental Fig. S2).These results suggest that miR-140 may suppress pathwaysother than Adamts-5 expression, thus regulating the over-all balance of cartilage matrix synthesis and degradation.Histone deacetylase 4 (HDAC4), which inhibits hypertro-phic differentiation of chondrocytes (Vega et al. 2004), isthought to be an miR-140 target gene (Tuddenham et al.2006); however, we did not observe significant changes inHDAC4 mRNA or protein expression in the endochondralplate or articular cartilage in the present study. The miR-140�/� and miR-140 TG lines may be useful in identifyingother targets associated with cartilage development andhomeostasis.






Taken together, our findings demonstrate that miR-140is required for skeletal development and cartilage homeo-stasis, and protects against OA-like pathology via Adamts-5 regulation (Fig. 7). We conclude that miR-140 is a novelregulator of cartilage homeostasis, and changes in its ex-pression and function play an important role in diseasesassociated with cartilage destruction, including OA andinflammatory arthropathies.

Materials and methods

Generation of miR-140-null mice and miR-140 TG mice

All animal experiments were performed according to protocolsapproved by the Institutional Animal Care and Use Committee atThe Scripps Research Institute and National Institute for ChildHealth and Development. A vector was constructed to replace theendogenous miR-140 locus with a PGK-neo cassette by homolo-gous recombination in embryonic stem (ES) cells. The 59 and 39

sequences flanking the endogenous miR-140 locus were amplifiedby PCR from a C57BL/6 genomic BAC clone (BACPAC ResourceCenter). These homologous arms were cloned into a vector incor-porating both a neomycin resistance cassette for positive selectionand a diphtheria toxin (DTA) gene for negative selection. The tar-geting vector was linearized and electroporated into TT2F mouseES cells. Recombinant ES clones were isolated after culture inmedium containing G418 antibiotic. Clones were then screenedfor proper integration by Southern blot analysis with the 59 probe,39 probe, and neomycin resistance cassette sequence indicatedin Figure 1. After proper integration was validated by genomicsequencing, two clones were chosen for microinjection into eight-cell-stage embryos. The resulting chimeric offspring were crossedwith C57BL/6 mice, and germline transmission was confirmedby Southern blot and PCR. The floxed PGK-neo cassette wasremoved by crossing with Meox-Cre TG mice; Cre-mediated neoexcision was confirmed by genomic PCR.

To generate cartilage-specific miR-140 TG mice, a pri-miR-140fragment was PCR-amplified from mouse chondrocyte cDNA withthe primers 59-TGGTGTGTGGTTCTATGCCAGC-39 and 59-AGCCTCAAGCCAGAATTCAGG-39.

pri-miR-140 was cloned into the NotI site of a Col2a1-basedexpression vector (Ueta et al. 2001), which contained the pro-moter and enhancer of the mouse Col2a1 gene. TG mice weregenerated by pronuclear injection of the transgene into the B6strain, and were backcrossed to a C57BL/6 background. GenomicDNA isolated from the tail was analyzed by PCR using specific

primers or transgene probes (forward primer, 59-CAGGCTGTGCTTGGTGGGCATCTG-39; reverse primer, 59-CTAGCCTAGGCTCGAGAAGCTTGCT-39).

qPCR

qPCR was performed using TaqMan Gene Expression Assay probesfor Adamts-5 (Mm01344182_m1) and glyceraldehyde 3-phosphatedehydrogenase (GAPDH; Mm 99999915_g1; Applied Biosys-tems), and qPCR for miR-140 was performed using the TaqManMicroRNA Reverse Transcription kit (Applied Biosystems)according to the manufacturer’s protocol. GAPDH or sno202was used as an internal control to normalize sample differences.

Western blot analysis

Total protein extracts of wild-type and miR-140�/� chondrocyteswere prepared for Western blot analysis. The Wwp2 protein,which is encoded by the host gene of miR-140, was detectedby the AIP2 antibody (dilution, 1:2000; sc-11896, Santa CruzBiotechnologies).

Radiologic imaging

X-ray and micro-CT imaging were performed using LaThetaLCT-200 (Aloka) according to the manufacturer’s instructions.Three-dimensional CT images of the knee joint were recreatedby using VGStudio MAX 2.0 software (Nihon Visual Science).

Bromodeoxyuridine (BrdU) labeling, RNA in situhybridization, and immunohistochemistry

Proliferating cells were detected by BrdU incorporation using theIn Situ Cell Proliferation kit, FLUOS (Roche); 49,6-diamidino-2-phenylindole (DAPI) served as a counterstain. In situ hybridi-zation of miR-140 was performed as described previously(Yokoyama et al. 2009). We used a probe specific for the primaryregion of miR-140. The sections of knee joints were immuno-stained using antibodies against ADAMTS-5 (ab13976, Abcam)and type II collagen (II-II6B3, Hybridoma Bank).

Histopathologic assessment

The miR-140�/� mice and wild-type littermates were obtainedfrom the intercross of miR-140+/� maintained in a C57BL/6background. Whole-mount Alcian blue and Alizarin red S stain-ing of skeletons were performed on wild-type and miR-140�/�

embryos at embryonic day 17.5 (E17.5). Mice were euthanized,and knee joints were harvested from 3-, 8-, and 12-mo-old mice.The knee joints were fixed in 10% zinc-buffered formalin (Z-Fix;Anatech) and decalcified in decalcifier (TBD-2; Shandon). Foreach animal, 4-mm sagittal sections through the central weight-bearing region of the medial femorotibial joint were stained withSafranin O and Fast Green for analysis of histopathologicdifferences between wild-type and miR-140�/� mice. The pro-teoglycan content of articular cartilage was scored using the pre-viously reported Mankin scoring system (Mankin 1971; Zemmyoet al. 2003). OA scores indicating the severity of cartilagedegeneration were evaluated for wild-type (n = 6) and miR-140�/� (n = 6) 3-mo-old mice, wild-type (n = 15) and miR-140�/

� (n = 13) 8-mo-old mice, and wild-type (n = 12) and miR-140�/� (n= 11) 12-mo-old mice. At least four sections per sample wereanalyzed microscopically and scored using previously reportedsemiquantitative scoring systems (Chambers et al. 2001; Glassonet al. 2004).

Figure 7. Adamts-5 regulation via miR-140 is required for skel-etal development and cartilage homeostasis. miR-140 has dualroles in both endochondral ossification and articular cartilagehomeostasis. During endochondral ossification, miR-140 is im-portant for chondrocyte proliferation, although its targets areunclear. miR-140 also plays a critical role in articular cartilagehomeostasis by repressing Adamts-5 expression.

Miyaki et al.





Mouse model of surgically induced OA

Surgical OA was induced in wild-type and miR-140�/�mice at theage of 10 wk by resecting the MMTL in the right knee joint(Glasson et al. 2007). Pathological changes in medial tibial plateauand medial femoral condyle of the joint were evaluated after 8 wkon safranin-O-stained sections by OA score.

Mouse model of AIA

Inflammatory arthritis was induced in knee joints of 10-wk-oldwild-type (n = 5), miR-140�/� (n = 6), and miR-140 TG (n = 6)mice by intra-articular injection of methylated bovine serumalbumin (mBSA; Sigma catalog no. A1009) in mice preimmu-nized with mBSA. On day 1, mice were preimmunized by a 100-mL intradermal injection at the base of the tail with an emulsioncontaining 100 mg of mBSA (in 0.9% saline) and an equal volumeof Freund’s complete adjuvant (Sigma catalog no. F5881). On day10, animals were treated with intra-articular mBSA (10 mL of 20mg/mL mBSA in 0.9% sterile saline or vehicle alone) into the leftand right knee joints. On day 17, the knee joints were harvestedand processed using standard procedures.

DNA microarray analysis

DNA microarray analysis was performed using the Affymetrixmouse genome 430 2.0 array. RNA samples were collected fromcultured rib chondrocytes of wild-type and miR-140�/� mice atP3. Microarray data were summarized by the Robust MultichipAverage (RMA) method, and statistical analysis was performedusing National Institute on Aging (NIA) Array Analysis (http://Igsun.grc.nia.nih.gov/ANOVA). The microarray data were de-posited in the Gene Expression Omnibus (GEO) repository underaccession number GSE16007.

Bioinformatics and miR-140 target analysis

We performed searches on the TargetScan prediction program(http://targetscan.org) for the predicted targets of miR-140.

Quantification of proteoglycan

Femoral heads were harvested from 4-wk-old wild-type and miR-140�/�mice. The explants were cultured for 3 d with or withoutIL-1b (5 ng/mL) in Dulbecco’s modified Eagle’s medium (DMEM)containing 10 mM HEPES and 1% penicillin/streptomycin. Atthe end of the culture, the cartilage explants were digested withprotease K solution (Tris-EDTA at pH 8.0, 30 mg/mL protease K,0.5% Tween 20) overnight at 50°C. Proteoglycan content in theprotease K digests and conditioned medium were determinedusing the Blyscan Glycosaminoglycan Assay kit (Biocolor).

Cell culture and transfection of ds-miR-140 assay

Mouse chondrocytes were prepared from rib cartilage of P3 miceby digestion with collagenase. Mouse chondrocytes were cul-tured in DMEM with 10% fetal bovine serum (FBS) at 37°C.dsRNA oligonucleotides (5 nM) representing mature sequencesthat mimic endogenous miR-140 and Silencer Negative ControlsiRNA #1 (Ambion) were transfected into chondrocytes withLipofectamine 2000 (Invitrogen). The synthesized RNA oligonu-cleotides 59-CAGUGGUUUUACCCUAUGGUAG-39 and 59-ACCACAGGGUAGAACCACGGAC-39 were annealed to obtainds-miR-140. Silencer Negative Control siRNA #1 (siNega) wasused at the same concentration as the specific miR-140 ds RNAin each experiment.

Luciferase assay

To create the pLuc2 reporter vector, a luciferase 2 gene (Promega)was incorporated into a modified pGL3-control plasmid withHindIII and EcoRI. To create the pGL3-miR-140 sensor vector(miR-140 sensor), the following chemically synthesized miR-140multiple target sites were annealed and inserted between EcoRIand XhoI sites downstream from the firefly luciferase codingregion in a pGL3 luciferase reporter plasmid with a modified39 UTR sequence (sense strand, 59-AATTCTACCATAGGGTAAAACCACTGCTACCATAGGGTAAAACCACTGCTACCATAGGGTAAAACCACTGCTACCATAGGGTAAAACCACTG-39; an-tisense strand, 59-TCGACAGTGGTTTTACCCTATGGTAGCAGTGGTTTTACCCTATGGTAGCAGTGGTTTTACCCTATGGTAGCAGTGGTTTTACCCTATGGTAG-39). To create the pLuc2-Adamts-5 39 UTR vector (Adamts-5 39 UTR), a fragment of the39 UTR of Adamts-5 gene including the predicted miR-140-bindingsite was PCR-amplified using the primer set 59-TGAATTCCTACTCTGCTTCCCTCTATGATC-39 and 59-TTCTCGAGCTGTGTTTCTTTCCTCAGGAG-39, and then cloned downstream from theluciferase 2 gene with EcoRI and XhoI. To create the reporter vectorwith mutated miR-140-binding site (Adamts-5 mut 39 UTR), the39 UTR of Adamts-5 was amplified with the primer set 59-TGAATTCCTACTCTGCTTCCCTCTATGATC-39 and 59-CCCTCTAGACATAGATTTGACGAATATCCGTTTACCCTCCTTAGG-39, andthen cloned into a Adamts5 39 UTR-containing vector with EcoRIand XbaI. ds-miR-140 and the scrambled siRNA sequences (AGUUGCACGCGCAAUUAdCdC and UAAUUGCGCGUGCAACUdTdT; final concentration, 50 nM) were reverse-transfected withLipofectamine RNAiMAX (Invitrogen) into HEK293T cells (2.0 3

104 cells per well in a 96-well plate). After 24 h of transfection,culture medium was changed, and the firefly luciferase reporterplasmid (20 ng) and Renilla luciferase control plasmid pRL-SV40 (20ng) were transfected with Lipofectamine 2000 (Invitrogen). Lucifer-ase activity was determined with the Dual-Glo Luciferase AssaySystem (Promega).

Statistical analysis

Two-tailed independent Student’s t-test and the nonparametricWilcoxon signed-rank test were used for statistical analysis.Asterisks indicate differences with statistical significance atP < 0.05 (*) and P < 0.01 (**).

Acknowledgments

We thank E. Kim for her excellent technical support, and extendour gratitude to all other members in the laboratory. We alsothank Y. Takahashi and M. Asada for their bioinformaticsassistance, and E. Lamar for critical reading of the manuscriptand discussion. T.S. and H.A. are associate scientists of TokyoMedical and Dental University. This project was supported byNIH AR050631 (to H.A.), AR056120 (to H.A.), AG007996 (toM.K.L.), and AG033409 (to M.K.L.); the Arthritis NationalResearch Foundation (S.M.); the Arthritis Foundation (S.O.);grants from the Ministry of Health, Labour, and Welfare; theGenome Network Project (MEXT); grants-in-aid for ScientificResearch (MEXT); and grants from National Institute of Bio-medical Innovation, Research on Child Health and Develop-ment, and The Japan Health Sciences Foundation.

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